Target SSR-Seq: A Novel SSR Genotyping Technology Associate With Perfect SSRs in Genetic Analysis of Cucumber Varieties

Abstract

Simple sequence repeats (SSR) - also known as microsatellites - have been used extensively in genetic analysis, fine mapping, quantitative trait locus (QTL) mapping, as well as marker-assisted selection (MAS) breeding and other techniques. Despite a plethora of studies reporting that perfect SSRs with stable motifs and flanking sequences are more efficient for genetic research, the lack of a high throughput technology for SSR genotyping has limited their use as genetic targets in many crops. In this study, we developed a technology called Target SSR-seq that combined the multiplexed amplification of perfect SSRs with high throughput sequencing. This method can genotype plenty of SSR loci in hundreds of samples with highly accurate results, due to the substantial coverage afforded by high throughput sequencing. We also detected 844 perfect SSRs based on 182 resequencing datasets in cucumber, of which 91 SSRs were selected for Target SSR-seq. Finally, 122 SSRs, including 31 SSRs for varieties identification, were used to genotype 382 key cucumber varieties readily available in Chinese markets using our Target SSR-seq method. Libraries of PCR products were constructed and then sequenced on the Illumina HiSeq X Ten platform. Bioinformatics analysis revealed that 111 filtered SSRs were accurately genotyped with an average coverage of 1289× at an extremely low cost; furthermore, 398 alleles were observed in 382 cucumber cultivars. Genetic analysis identified four populations: northern China type, southern China type, European type, and Xishuangbanna type. Moreover, we acquired a set of 16 core SSRs for the identification of 382 cucumber varieties, of which 42 were isolated as backbone cucumber varieties. This study demonstrated that Target SSR-seq is a novel and efficient method for genetic research.

Keywords: DNA fingerprint; cucumber; genetic diversity; simple sequence repeat; target SSR-seq.

FIGURE 1

Target SSR-seq pipeline. Schematic workflow of perfect SSR selection, Multiplexed PCR design, high-throughput sequencing, and authentic SSR genotype.

FIGURE 2

Target SSR-seq genotyping result analysis. The distribution of reads alignment (A), target region alignment (B), average read depths (C), and uniformity index (D) for 382 cucumber varieties.

FIGURE 3

Genetic characterization of 111 SSRs in 382 cucumber varieties. (A) Distribution of 111 SSR loci in seven cucumber chromosomes. 16 core SSR set is labeled in red. (B) Allele numbers per SSR locus. (C) Observed heterozygosity. (D) Genetic diversity. (E) PIC value.

FIGURE 4

Population structure of 382 cucumber varieties. (A) Delta K plots derived from Target SSR-seq result. (B) Two populations were observed in 382 varieties, Pop1 is colored in pink and Pop2 is colored in green. (C) Four subpopulations were classified and Pop1A, Pop1B, Pop2A, and Pop2B are colored with red, blue, yellow, and green, respectively.

FIGURE 5

Unrooted neighbor-joining tree of 382 cucumber varieties. The Pop1A, Pop1B, Pop2A, and Pop2B subgroups are colored the same as in Figure 4.

FIGURE 6

Principal co-ordinates analysis (PCoA) of 382 cucumber varieties. The Pop1 and Pop2 are labeled with circle and triangle. The four subpopulations are colored the same as in Figure 4.

FIGURE 7

The saturation curve of 111 SSRs identifying in 382 cucumber varieties. A total of 16 SSRs identified 99% cucumber varieties.